Peroxisome biogenesis disorders (PBDs) are inborn errors of metabolism with considerable clinical, biochemical, and genetic heterogeneity. The rational design of effective treatments for these disorders requires the precise identification of the causative molecular defects in each PBD patient. However, the diagnosis of PBD patients is time consuming, labor intensive, and highly specialized since it requires establishing fibroblast cultures and performing biochemical lipid analyses for every individual tested. Our goal is to rapidly and efficiently determine the genetic basis for PBDs in order to improve the likelihood of identifying and developing therapies that are most effective for each patient. We hypothesize that high-throughput mutation detection and gene expression assays can rapidly and efficiently diagnose the molecular basis for PBDs. To test our hypothesis, we will identify causative mutations in genomic DNA from patients within the Zellweger syndrome-neonatal adrenoleukodystrophy-infantile Refsum disease (ZS-NALDIRD) spectrum of PBDs and analyze their activity in cultured fibroblasts from patients with known mutations. In this proposal, we will develop novel oligonucleotide microarray-based diagnostics to rapidly and efficiently screen genomic DNA from patient blood samples for mutations in five genes (PEX1, PEX6, PEX10, PEX12, and PEX26) which account for over 90% of ZS-NALD-IRD cases (Specific Aim 1). We will determine the specificity and sensitivity of these assays by blinded mutational analyses of ZS-NALD-IRD patients with known mutations in these genes. After this validation step, we will use these microarray assays to uncover the genetic defects responsible for clinical phenotypes in one hundred ZS-NALD-IRD patients. Furthermore, we will conduct functional assays to determine the functional significance of missense mutations in order to better correlate patient genotype with clinical phenotypes (Specific Aim 2). First, we will prioritize missense changes for analysis based on the conservation of these amino acids in other species and allele frequencies in the general population. Afterwards, we will construct lentiviral vectors that can transduce wild type and mutated PEX genes into PEX1, PEX6, PEX10, PEX12, and PEX26 null cell lines that lack functional peroxisomes due to defects in peroxisome assembly. We will determine the functional activity of PEX genes containing missense changes relative to their wild type counterparts by screening for the appearance of peroxisomes in the appropriately transduced null cell lines. Overall, these mutation detection (Specific Aim 1) and functional (Specific Aim 2) assays will provide valuable resources for the precise molecular diagnosis and characterization of patients within the ZS-NALD-IRD spectrum. In addition to providing the genetic information needed for the rational design of therapies, these high-throughput genetic tests will allow for more accurate predictions of clinical outcomes for these individuals and facilitate prenatal diagnosis in future pregnancies. [unreadable] [unreadable]